Quantitative polymerase chain reaction (qPCR) is used to simultaneously detect a specific DNA sequence in a sample and determine the actual copy number of this sequence relative to a standard. In real-time PCR, the DNA copy number can be established after each cycle of amplification. This is useful for diagnostic tests where the cycle number at which the signal of the reaction crosses a threshold (threshold cycle) has been correlated with clinical data (e.g., growth of an organism in culture, or the presence of marker genes from metastatic cancer cells in a sentinel lymph node). And by beginning with a reverse transcription step, qPCR can be used to quantify numbers of messenger RNAs as well. There are many applications for qPCR in both research and diagnostics. BioTrove's Open Array DLP real-time qPCR platform (Woburn, MA, USA) is a high-density platform that can be used with TaqMan chemistries to perform more than 9,000 real-time PCR reactions simultaneously. Each plate has 3078 “through-holes,” the equivalent of eight 384-well plates, and up to 3 plates can be cycled and imaged in the OpenArray NT Cycler at one time. This platform can be used for detecting both presence and types of microorganisms as well as for validating microarray results, among other applications, in nanoliter sample volumes. Two investigators who apply this platform in their research discuss their work.
Changing Clinical MicrobiologyRobert Slinger, Program Director, Medical Microbiology/Pediatric Infectious Diseases, Children's Hospital of Eastern Ontario (CHEO), and Assistant Professor, University of Ottawa, Ontario, Canada, is interested in identifying multiple pathogens quickly and simultaneously in a variety of patient specimens. “We wanted to see if we could use qPCR to figure out the cause of infections, and thought the BioTrove platform had the most potential because we could do thousands of reactions at once,” he says. Slinger explains that rapid assays capable of detecting dozens of infectious agents simultaneously will help improve patient management and allow physicians to choose the correct antibiotic or antiviral agent, or, alternatively, not to prescribe any unnecessary agents.
Slinger's group used group A Streptococcus (GAS) infections for proof-of-concept studies, demonstrating that they could detect the organism on throat swab samples using qPCR with the sensitivity and specificity of culturing the organism in vitro, the reference (“gold”) standard.
They also examined the ability of the platform to detect respiratory syncytical virus (RSV), which Slinger says is a leading cause of pediatric hospital admission, and have been able to identify the virus with the same sensitivity using nanoliter PCR as the reference standard for RSV, which is fluorescent antibody detection. In this instance, primers for the RSV N (nucleo-protein) and L (polymerase) genes were used.
Slinger's group has created a panel representing over 30 types of both bacteria and viruses associated with respiratory infections in children and with community-acquired pneumonia in adults. These will be tested on nasal, throat, and sputum samples. “It's attractive to incorporate all of these and test for them at the same time,” he observes. His group has already shown that multiplex nanoliter qPCR, which allows for the identifcation of different organisms via their nucleic acid sequences, is feasible on nasal samples from children with coughing and difficulty breathing.
“I think this will be cost-effective in a hospital setting, although it's still in the research setting,” Slinger says. The elimination of the different tests and costly culture techniques could contribute to savings. Time savings in the analyses are important, too. Slinger says that in their hands, the current qPCR technology requires about four hours to obtain a result, which he thinks could even be a little shorter in the future. “As a clinical person, I would like it to get shorter,” he says.
Analyzing the EnvironmentSyed A. Hashsham, Associate Professor, College of Engineering, Michigan State University, Lansing, Michigan, is interested in being able to detect a large number of genes from pathogenic organisms, or the organisms themselves from a large number of samples. A common technical goal among these projects is to be able to measure a number of genes or organisms within a complex system (i.e., water or soil containing considerable amounts of DNA from unknown sources), without having to isolate and culture the organisms first. Hashsham says that in general, his work requires amplification of the target gene before microarrays can be used in a detection step, and that his group is studying samples containing up to a few dozen targets along with analyses that involve hundreds of samples. For example, there are about 20 waterborne pathogens Hashsham's group is interested in studying. In the past, detecting these pathogens in various samples had been performed one organism at a time. But Hashsham's group is developing plates to analyze about 100 samples for as many as 500 DNA markers in parallel which will be used to distinguish between human and animal pathogens. He points out that for success, this approach will need to be validated.
Although Hashsham says that the Environmental Protection Agency (EPA) probably wouldn't approve the routine use of this kind of test because of the expense, he suspects it might prove useful for investigating outbreaks of waterborne infections. “We do need safe water,” he says, “but it will not be through regulation. It's too expensive.” His group is working on ways to measure more organisms less expensively in multiple water samples. Another issue is the variation in water regulations among communities and what they are required to report.
Antibiotics, when used in large quantities for raising farm animals commercially, can contaminate the watershed: as a result, the surrounding microbial community can develop resistant genes, which can be tracked through the environment. “You need to look at the background,” Hashsham notes, to see what and how many antibiotic-resistant genes are being carried by animals when given antibiotic-free feed versus antibiotic-containing feed. Hashsham's group is working on determining the presence of antibiotic resistance genes in environmental samples (e.g., from farms), and identifying specific genes in samples (e.g., those that might be useful in biore-mediation) without having to know which organisms are in those samples.
Future Directions“My idea of the future is ‘ABC,’ amplify before culture,” says Slinger. “qPCR can be used to amplify and see what's in the sample. The positives [i.e, organisms from positive results] could be cultured after, if desired. Most respiratory viruses don't grow well in culture, so samples could be cultured selectively after qPCR.” He also points out that doing a rapid test first allows infected patients to be treated appropriately and sooner.
Future directions include initiatives on safe food and water. One current study conducted by Slinger and colleagues at CHEO, Nunavut Department of Health, Qikiqtani General Hospital, Nunavut, and Food Directorate, Health Canada, and funded by the Canadian Institutes for Health Research, is investigating food-and waterborne infections in Canadian Northern Aboriginal communities. This study will look at parasites in addition to bacteria and viruses. Some of the causative organisms (e.g., norovirus) are difficult to detect in stool samples. This study will demonstrate the wider application of the qPCR approach to pathogen detection. Other potential panels containing multiple targets could include organisms responsible for sexually transmitted infections, blood-stream infections, meningitis, and tropical diseases.
qPCR could also enable the detection of H1N1 influenza A virus, for which a rapid test is desirable; the current antibody tests for this strain are not as sensitive as those for other strains. Additionally, a test for another influenza strain, H5N1, could be used during suspected outbreaks. Detection of antibiotic resistance, as Hashsham observes, is an important application, too. “We have plans to look for antibiotic resistance genes in Gram-negative bacteria on the drawing board,” says Slinger. His group would also like to see if they can detect organisms that are notoriously slow-growing from sputum samples (e.g., Mycobacterium tuberculosis and Legionella).
“Another point to make about qPCR,” Slinger says, “is that publications have said that for some respiratory viruses, the quantity of virus predicts how sick the patient will be. Viral load is important with human immunodeficiency virus (HIV). For some of the respiratory viruses, including RSV, we are going to ask, ‘is the viral load helpful clinically?’” He notes that in a clinical setting, qPCR could provide more quantitative information to physicians when treating their patients. “The quantitative side is an advantage.”
